The underlying causes of diseases associated with smooth muscle cannot be understood until the events that account for contractions in response to excitatory neurotransmitters such as norepinephrine and acetylcholine are known. Several lines of evidence suggest that specific alterations in phospholipids may mediate cholinergic actions on contractile state. We have postulated that the accumulation of phosphatidic acid or metabolically related lipids mediate the contraction of smooth muscle in response to cholinergic stimulation. Studies to evaluate this hypothesis will be conducted in isolated smooth muscle cells prepared from toad stomach, a preparation that affords several advantages for studies of the physiology and biochemistry of smooth muscle. To evaluate the hypothesis studies will determine the nature and manner by which cholinergic stimulation alters phospholipid metabolism. This information will then be used to identify conditions and agents which amplify, attenuate or mimic these changes and determine whether these conditions produce like changes in the contractile response. Studies using microelectrode recording will determine whether cholinergic stimulation produces contraction by membrane depolarization and/or mechanisms independent of membrane voltage. Studies using Ca2+ sensitive microelectrodes will establish whether cholinergic stimuli increase intracellular Ca2+ and whether this increase is a sole consequence of membrane depolarization. Studies will then determine whether accumulation of phosphatidic acid accounts for the cholinergic effects on intracellular Ca2+. It is anticipated that performance of these studies will provide significant information on the physiological and biochemical events associated with cholinergic action in smooth muscle. The results from these studies may well have significant impact on the manner by which a variety of hormones and alter agents after intracellular Ca2+ in a variety of other cell types.